The present invention relates to a recombinant herpes simplex virus (HSV), especially type 1 (HSV-1) or type 2 (HSV-2) having a good ability to continuously express an inserted heterologous gene whilst the virus is at the same time maintained in its latent non-replicative state.
A distinguishing feature of herpes virus infections is the ability to persist in the host for long periods in a non-replicative or latent state. Herpes simplex virus type 1 (HSV-1) establishes latent infection in human peripheral sensory ganglia and can reactivate to produce recurrent mucocutaneous lesions. Operationally, the pathogenesis of herpes virus infections can be divided into several distinct stages which can be studied individually in experimental animal models: acute viral replication, establishment of latency, maintenance, and reactivation. Following inoculation, HSV-1 replicates at the site of inoculation and is transported to sensory ganglia. Replication at the periphery or in sensory ganglia may increase the amount of virus that can establish latent infection. During latent infection, HSV-1 DNA can be detected in infected tissues but infectious virus cannot be detected. This latent state is often maintained for the life of the host. A variety of stimulae (such as fibrile illness and exposure to ultraviolet irradiation) can interrupt the latent state and cause the reappearance of infectious virus or reactivation.
Transcription of the HSV-1 immediate early (IE) genes is not detectable during latency. However, in tissue culture, IE gene expression is a pre-requisite for viral replication. Transcription of the IE genes is transinduced by a virion protein Vmw65 (transinducing factor) that is a component of the HSV-1 virion. Vmw65 does not bind directly to HSV-1 DNA but mediates transinduction by association with cellular proteins to form a complex which interacts with the IE regulatory element.
Ace et al (1989) report an HSV-1 mutant which contains a 12 bp insertion in the coding region of Vmw65 which is unable to transinduce IE gene expression, though the altered Vmw65 is incorporated into mature virions.
The inventor""s previous patent specification WO91/02788 discloses a herpes simplex virus type 1 mutant which includes the mutation within the Vmw65 gene which removes the transinducing properties of the Vmw65 transactivator protein such that the virus remains in its latent state. In addition, a xcex2-galactosidase marker gene under the control of the latency associated transcript (LAT) promoter is inserted into the thymidine kinase (TK) gene and expression of the heterologous gene during latency is observed.
It is an object of the present invention to provide an HSV viral vector having enhanced expression of the inserted heterologous gene during latency.
Generally speaking, the present invention is based on the discovery that enhanced long term expression during latency may be obtained by use of the IE1 gene enhancer of cytomegalovirus controlling the inserted heterologous gene.
Most specifically, the present invention provides a recombinant herpes simplex virus (HSV) viral vector genome which comprises;
(i) a DNA sequence change in the gene coding for Vmw65 protein such as to substantially remove transinducing properties thereof; and
(ii) an expressable heterologous gene inserted into a region of the HSV genome which is non-essential for culture of the virus, the gene being under the control of the immediate early 1 (IE1) gene enhancer of cytomegalovirus (CMV).
The Vmw65 sequence change removes the transinducing properties thereof such that expression of HSV IE genes and therefore HSV viral replication in vivo, is substantially removed. The HSV vector is therefore constrained to remain in its latent state. The use of the IE1 CMV enhancer to control the inserted heterologous gene has been found to give excellent long term expression of the heterologous gene during latency. Experiments in mice using the inserted heterologous lacZ gene have showed continuous expression from the latent vector of up to five months. In contrast, use of other promoters such as HSV-1 Vmw110 and Vmw65, and the Moloney murine leukaemia virus enhancer have been found not to give long term expression during latency.
The structure of the human cytomegalovirus (HCMV) enhancer is discussed in Stinski and Roehr (1985). The IE1 enhancer is the promoter-regulatory region upstream of the major immediate early gene of human cytomegalovirus. This enhancer region upstream of the IE1 gene consists of a series of different repeat sequences distributed up to xe2x88x92509 bp from the site for the initiation of transcription. Within this enhancer are a set of inducing sequences. Certain of the sequences within the enhancer region are non-essential and do not effect the level of expression obtained, whilst other sequences promote downstream expression.
The CMV enhancer is generally that derived from human cytomegalovirus (HCMV) and the immediate early 1 (IE1) nomenclature applies particularly to that virus. However, the analogous enhancer from other types of CMV, such as mouse, rat, equine, simian, and guinea pig CMV, may also be employed.
The present invention primarily envisages the use of the entire CMV IE1 enhancer sequence. Indeed in a preferred embodiment of the invention a larger sequence extending to xe2x88x92730 bp and including the entire CMV IE1 enhancer was employed. However, it is clearly within the ambit of the skilled man to modify the naturally occurring enhancer sequence without departing from the general scope of the present invention. Thus, the present invention is concerned not only with the use of the entire CMV IE1 enhancer sequence but also with variations in that sequence, either by insertion, deletion or substitution such that the enhancer properties are not substantially affected.
Other promoter sequences, such as the LAT (latency associated transcript) promoter, may be included upstream of the inserted heterologous gene and HMCV enhancer, but these have not been found to offer any particular advantage according to the present invention.
The position and size of the DNA sequence change in the gene coding for Vmw65 protein is significant, since it is necessary to substantially remove the transinducing properties of the Vmw65 protein (and thereby prevent in vivo replication of the virus and consequent illness of the patient), whilst at the same time retaining the structural properties of the protein required to successfully assemble the complete virion when the virus is cultured. The viral vector of the present invention must be capable of replication under culture conditions so as to be able to produce sufficient quantities of the mutant virus for use, but at the same time the virus should be incapable of replication in vivo. Preferably, the DNA sequence change is achieved by a transition (purine to purine or pyrimidine to pyrimidine) or transversion (purine to pyrimidine or vice versa) alteration of 1-72 base pairs, an oligonucleotide insert of 3-72 base pairs or a deletion of 3-72 base pairs, at a position between amino acids 289 and 480 (especially 289 and 412) of the Vmw65 protein.
The recombinant HSV may be of type HSV-1 or HSV-2 or may be an intertype recombinant between HSV-1 and HSV-2 which comprises nucleotide sequences derived from both types. The recombinant HSV genome will generally be contained in a mutant HSV virus.
HSV has the ability to infect many tissue types and therefore in principle the viral vector of the present invention may be used as a vector directed against a wide variety of cell types. Latency in HSV infection tends to be established within neuronal cells, though it is possible that expressed gene products may translocate from their original point of production. The viral vector of the present invention is thus particularly useful for delivering expressable heterologous genes into neuronal cells. The genes may deliver a therapeutic effect or may deliver an antigenic protein for stimulating the production of antibodies when used as a vaccine. The therapeutic gene is generally a gene associated with a neurological genetic deficiency i.e. it compensates for an inherited or acquired genetic deficiency. Examples of such therapeutic genes include:
(a) human, rat or mouse tyrosine hydroxylase genes 1, 2 or 3, which are relevant to the alleviation of symptoms of Parkinson""s disease;
(b) human, rat or mouse nerve growth factor (e.g. the beta subunit) for treatment of Alzheimer""s disease and Parkinson""s disease;
(c) human, rat or mouse hypoxanthine-guanine phosphoribosyl transferase gene for the treatment of Lesch-Nyhan disease;
(d) human beta-hexosaminidase alpha chain gene, for the treatment of Tay-Sachs and Sandhoff""s diseases; and
(e) human immunodeficiency virus (HIV) nef gene, for the control of neurological symptoms in HIV-positive individuals.
In particular, the in situ expression of tyrosine hydroxylase by the HSV viral vector of the present invention may help alleviate the symptoms of Parkinson""s disease. Tyrosine hydroxylase is a crucial enzyme in the synthesis of dopamine. Deficiency of dopamine is the major cause of symptoms in Parkinson""s disease, and current treatment involving the administration of L-dopa gives onlyxe2x80x94short-lived respite.
The heterologous gene may be inserted into any region of the viral genome which is non-essential for the culture of the virus, i.e. replication of the virus outside the body, particularly in tissue culture. In particular, the insertion of the heterologous gene could be made in the coding sequences or in the flanking control regions of one or more of the following HSV-1 genes:
1. The thymidine kinase gene (the UL23 gene); which is the preferred choice since thymidine kinase is important for pathogenicity of HSV, so that deactivation of its gene may reduce potential pathogenicity of the mutant vector.
2. The RL1 gene
3. The RL2 gene (otherwise named the IE110 gene)
4. The locus encoding the latency associated transcripts
5. The UL2 gene (otherwise named the Uracil-DNA glycosylase gene)
6. The UL3 gene
7. The UL4 gene
8. The UL10 gene
9. The UL11 gene
10. The UL13 gene
11. The UL16 gene
12. The UL20 gene
13. The UL24 gene
14. The UL40 gene (otherwise named the gene encoding the small subunit of ribonucleotide reductase)
15. The UL41 gene (otherwise named the virion host shutoff factor gene)
16. The UL43 gene
17. The UL44 gene
18. The UL45 gene
19. The UL46 gene
20. The UL47 gene
21. The UL50 gene (otherwise named the dUTPase gene)
22. The UL55 gene
23. The UL56 gene
24. The US1 gene (otherwise named the IE68 gene)
25. The US2 gene
26. The US3 gene (otherwise named the protein kinase gene)
27. The US4 gene (otherwise named the glycoprotein G gene)
28. The US5 gene
29. The US7 gene (otherwise named the glycoprotein I gene)
30. The US8 gene (otherwise named the glycoprotein E gene)
31. The US9 gene
32. The US10 gene
33. The US11 gene
34. The US12 gene (otherwise named the IE12 gene)
The UL, US and RL nomenclature system given above is a systematic one, but certain common names of genes are also included.
Another aspect of the present invention relates to the use of the recombinant HSV viral vector genome comprising an appropriate expressible therapeutic gene in the therapy of disease, particularly diseases due to or associated with genetic deficiency. The viral vector may also be used as a vaccine to deliver an antigenic protein.
A further aspect of the present invention relates to a pharmaceutical composition for administering the viral vector comprising the viral vector in admixture with a pharmaceutically acceptable carrier. Generally, the composition will be formulated for parenteral administrationxe2x80x94usually by injectionxe2x80x94in an appropriate acceptable carrier such as apyrogenic isotonic saline.
The present invention is hereafter further described by way of example only, the insertion of a gene (the lacZ gene coding for xcex2-galactosidase) into the viral vector of the present invention. The lacZ gene is inserted in order to demonstrate the technology, since the presence of the gene is easily detectable. However, for therapeutic or other applications, a heterologous gene would be inserted in an analogous manner; or the lacZ gene could be directly replaced by another heterologous gene.